Development of a one-step ELISA method using an affinity peptide tag specific to a hydrophilic polystyrene surface

Glutathione S-transferase genetically fused with an affinity peptide tag, PS19 (RAFIASRRIKRP) having a specific affinity for a hydrophilic polystyrene (PS) surface, was preferentially immobilized on a hydrophilic PS (phi-PS) plate without suffering from interference by coexisting protein molecules. Furthermore, rabbit IgG chemically conjugated with a peptide, KPS19R10, in which (10)Lys in PS19 was replaced with Arg and one Lys residue was added at the N-terminus as a coupling site for glutaraldehyde, showed a higher immobilization affinity to the phi-PS plate than that conjugated with the PS19 peptide. On the basis of these findings, the use of a phi-PS plate and peptide tag-linked ligand proteins permitted a one-step or two-step enzyme-linked immunosorbent assay (ELISA) to be achieved, resulting in a substantial reduction in operational time compared with the conventional ELISA method using a hydrophobic PS (pho-PS) plate, while maintaining a high sensitivity. Furthermore, the sensitivity was increased to a greater extent compared to the conventional ELISA meihod when the one-step ELISA was applied to the detection of bovine insulin in a sandwich mode, due to the reduced number of washing and incubation steps. The method proposed here would be a versatile method for use in various ELISA techniques such as sandwich and competitive ELISAs using an antigen, an antibody and streptavidin that are genetically fused or chemically conjugated with the PS-specific affinity peptide as the ligand protein.     

Immobilization strategies for single-chain antibody microarrays

Sandwich ELISA microarrays have great potential for validating disease biomarkers. Each ELISA relies on robust-affinity reagents that retain activity when immobilized on a solid surface or when labeled for detection. Single-chain antibodies (scFv) are affinity reagents that have greater potential for high-throughput production than traditional IgG. Unfortunately, scFv are typically less active than IgG following immobilization on a solid surface and not always suitable for use in sandwich ELISAs. We therefore investigated different immobilization strategies and scFv constructs to determine a more robust strategy for using scFv as ELISA reagents. Two promising strategies emerged from these studies: (i) the precapture of epitope-tagged scFv using an antiepitope antibody and (ii) the direct printing of a thioredoxin (TRX)/scFv fusion protein on glass slides. Both strategies improved the stability of immobilized scFv and increased the sensitivity of the scFv ELISA microarray assays, although the antiepitope precapture method introduced a risk of reagent transfer. Using the direct printing method, we show that scFv against prostate-specific antigen (PSA) are highly specific when tested against 21 different IgG-based assays. In addition, the scFv microarray PSA assay gave comparable quantitative results (R(2) = 0.95) to a commercial 96-well ELISA when tested using human serum samples. In addition, we find that TRX-scFv fusions against epidermal growth factor and toxin X have good LOD. Overall, these results suggest that minor modifications of the scFv construct are sufficient to produce reagents that are suitable for use in multiplex assay systems.     

Site-directed immobilization of antibody onto solid surfaces for the construction of immunochip

The performance of an immuno-analytical system can be assessed in terms of its analytical sensitivity,i.e., the detection limit of an analyte, which is determined by the amount of analyte molecules bound to the capture antibody that has been immobilized onto a solid surface. To increase the number of the binding complexes, we have investigated a site-directed immobilization of an antibody that has the ability to resolve a current problem associated with a random arrangement of the insolubilized immunoglobulin. The binding molecules were chemically reduced to produce thiol groups that were limited at the hinge region, and then, the reduced products were coupled to biotin. This biotinylated antibody was bound to a streptavidincoated surface via the streptavidin-biotin reaction. This method can control the orientation of the antibody molecules present on a solid surface and also can significantly reduce the possibility of steric hindrance in the antigen-antibody reactions. In a two-site immunoassay, the introduction of the site-directly immobilized antibody as the capture enhanced the sensitivity of analyte detection approximately 10 times compared to that of the antibody randomly coupled to biotin. Such a novel approach would offer a protocol of antibody immobilization in order for the possibility of constructing a high performance immunochip.     

Development and application of competitive ELISA assays for rat LH and FSH

Rat LH (rLH) and FSH (rFSH) were measured by sensitive and specific competition ELISAs. The rat LH ELISA used rLH-I-9 coated plates, an antiserum against rLH and an antibody against rabbit IgG labeled with peroxidase. Using rLH-RP-3 as a standard, rat LH was determined by binding of the anti-LH antibody to rLH-I-9 coated plates. The sensitivity of the assay was 0.8 ng/mL. Similarly, the rat FSH-ELISA used rFSH-I-8 coated plates, an antiserum against rFSH and an antibody against rabbit IgG labeled with peroxidase. Using rFSH-RP-3 as a standard, the FSH-ELISA was also determined by binding of the anti-FSH antibody to rFSH-I-8 coated plates. The sensitivity of this assay was 1.25 ng/mL. Both rat LH and FSH ELISA assays are highly specific and provide accurate determination of gonadotrophins in buffers, sera, cell culture media, and anterior pituitary extracts. These assays were used for monitoring the gonadotrophin surge-attenuating factor (GnSAF) and inhibin activities present in human follicular fluid (hFF). The 2 new ELISA procedures have practical advantages (safety, convenience, economy) over the RIA methods, and they perform as well as the RIA techniques at the same range of concentrations.     

Autodisplay of 60-kDa Ro/SS-A antigen and development of a surface display enzyme-linked immunosorbent assay for systemic lupus erythematosus patient sera screening

Enzyme-linked immunosorbent assay (ELISA) is a common tool to test human sera on an antibody reaction against a specific antigen. The 60-kDa Ro/SS-A antigen for autoantibodies can be found in sera from systemic lupus erythematosus (SLE) patients. As in the case of 60-kDa Ro/SS-A, antigens used in ELISAs are recombinantly expressed in Escherichia coli and time-consuming purification steps are needed to get the proteins. To avoid these disadvantages, 60-kDa Ro/SS-A was expressed on the surface of E. coli using autodisplay, an efficient surface display system. Cells displaying 60-kDa Ro/SS-A on the surface were applied as an antigen source instead of the purified antigen. In total, 39 patients and 30 control sera were screened on a 60-kDa Ro/SS-A antibody reaction. To eliminate antibodies against native E. coli, human sera were preabsorbed with E. coli cells prior to the assay. The new ELISA protocol (surface display ELISA [SD-ELISA]) using E. coli with autodisplayed 60-kDa Ro/SS-A showed a sensitivity of 86.67% and a specificity of 83.33% by a cutoff value of 0.28. Our results show that autodisplay provides simple, rapid, and cheap access to human antigens for an ELISA to screen human sera against specific antibody reactions.     

Development and Evaluation of Capture Enzyme-Linked Immunosorbent Assays for Detection of Immunoglobulin G and M Antibodies to Group A Streptococcal Antigens

Capture enzyme-linked immunosorbent assays (ELISAs) were developed to detect immunoglobulin G and M antibodies to group A streptococcal (GAS) antigens, streptolysin O, streptokinase, and group A carbohydrate. The sensitivities and the specificities of the IgM capture ELISAs to each GAS antigen were high enough to distinguish the patients with GAS infections (diagnosed as GAS pharyngitis or scarlet fever) from the control groups (healthy people and patients with pharyngitis from whom GAS could not be isolated). On the other hand, the specificities of the IgG capture ELISAs were not very effective in diagnosis of GAS infections. When the capture ELISA and an indirect ELISA detecting IgM antibodies to group A carbohydrate were compared, false-positive reactions due to rheumatoid factor occurred in the indirect ELISA, but did not occur in the capture ELISA. These results indicate that the capture ELISA works better than the indirect ELISA in detecting the IgM antibody, and that the IgM capture ELISA to GAS antigen provides a rapid and highly reliable serodiagnosis for GAS infections employing only a single serum.     

The repetitive use of samples to measure multiple cytokines: the sequential ELISA

The inexpensive and highly effective enzyme-linked immunosorbant assay (ELISA) is widely used for the quantification of biomarkers in a variety of biological samples. The applicability of the standard ELISA is difficult when experiments yield low volume samples. In such studies, the capacity of sample collection system does not meet the sample volume requirements to measure multiple different cytokines by the traditional ELISA protocol. In the modified methodology of the sequential ELISA, samples are re-used in multiple successive cycles, dramatically increasing the number of biomarkers which may be measured. Although the protocols presented to date were developed for quantification of cytokines in either blood plasma or cerebrospinal fluid, the sequential ELISA protocol has wide potential for further uses. When only limited quantities of samples are available for analysis, the sequential ELISA technique based on commercially available antibody pairs can be an attractive alternative to more advanced, costly multiplex methods. Additionally, any laboratory that currently runs traditional ELISAs has all the necessary equipment and reagents to perform the sequential ELISA.     

Development of surface-based assays for transmembrane proteins: selective immobilization of functional CCR5, a G protein-coupled receptor

A general method to develop surface-based assays for transmembrane (TM) receptor function(s) without the need to isolate, purify, and reconstitute the proteins is presented. Based on the formation of an active surface that selectively immobilizes membrane vesicles, the method is illustrated using the chemokine receptor CCR5, a member of the largest family of cell surface eukaryotic TM proteins, the G protein-coupled receptors (GPCRs). The method begins with a protein-resistant surface containing a low percentage (1-5%) of surface-bound biotin on gold as the initial template. Surface plasmon resonance (SPR) data show specific immobilization of functional CCR5 after the initial template is activated by immobilization of rho 1D4 antibody, an anti-rhodopsin monoclonal antibody specific for the carboxyl terminal nine amino acids on bovine rhodopsin that had been engineered into the carboxyl terminus of CCR5, and exposure to vesicles obtained from mammalian cells transfected with a synthetic human CCR5 gene. Activation of the initial template is effected by sequential immobilization of avidin, which binds to the biotin in the initial template, a biotinylated goat anti-mouse immunoglobulin G (Bt-IgG), which binds to the avidin binding sites distal to the surface and the F(c) portion of the rho 1D4 antibody through its F(ab) region(s) and finally rho 1D4. This approach establishes a broad outline for the development and application of various assays for CCR5 functions. SPR data also showed that vesicle immobilization could be achieved through an integrin-integrin antibody interaction after activation of the initial template with a goat anti-human integrin beta1 antibody. These results suggest that the generic nature of the initial platform and flexibility of the subsequent surface activation for specific immobilization of membrane vesicles can be applied to the development of assays for other GPCRs or TM receptors for which antibodies are available or can be engineered to contain a particular antibody epitope.     

Triple-site antigen capture ELISA for human myoglobin can be more effective than double-site assay

Using a panel of monoclonal antibodies against human myoglobin (Mb), we have shown that the sensitivity of antigen-capture ELISA can be significantly increased by simultaneous immobilization of two cooperating capture monoclonal antibodies on a solid phase. This method ("triple-site ELISA") uses three monoclonal antibodies to different epitopes of the same antigen (two capture/one tracer) unlike the traditional double-site assay using one capture and one tracer monoclonal antibody. We developed double- and triple-site ELISA for Mb by varying the capture and tracer monoclonal antibodies. Triple-site assays showed 4-6-fold increase in sensitivity compared to the double-site assays. A model for this effect is suggested; according to the model, in triple-site ELISA, high-affinity cyclic configurations can be formed by an antigen, two capture monoclonal antibodies, and the surface of the solid phase.     

A competitive enzyme-linked immunosorbent assay: applications in the assay of peptides, steroids, and cyclic nucleotides

Indirect competitive enzyme-linked immunosorbent assays (ELISAs) that can be used to quantify several types of small, bioactive molecules, including peptides, steroids, and cyclic nucleotides, are described. The assays require no special expertise to perform, and the sensitivities are very high, equally or exceeding what is commonly achieved in radioimmunoassay (RIA). The molecule to be assayed or a synthetic derivative is coupled to a protein carrier (= conjugate). The conjugate is adsorbed to the wells of a microtiter plate where it is bound by antibody in inverse proportion to free hapten in a sample or standard. Bound antibody is then quantified with enzyme-labeled anti-immunoglobulin and appropriate substrate. The assay of peptides is illustrated for the sulfated cholecystokinin octapeptide, in which an ED50 of 20 fmol (2 x 10(-10) M in 100 microliters assay volume) is attained. The ED50's and slopes of the dose-response curves in the steroid and cyclic nucleotide ELISAs are compared with those parameters obtained earlier by RIA using the same antisera. This comparison indicates that a steroid, ecdysone, can be quantified with no apparent participation of the bridging group of the conjugate in the competitive assay. Furthermore, the ED50's in the ecdysone assays (ecdysone 2 beta, 3 beta, 14 alpha, 22R, 25-pentahydroxy-5 beta-cholest-7-en-6-one, 7.7 fmol; 20-hydroxyecdysone, 16 fmol) are 19- to 38-fold lower for ELISA than for RIA. In the cyclic nucleotide assay, the bridge of a cAMP conjugate (homologous with the bridge of the immunogen) decreases the slope of the dose-response curve. This effect is minimized by the use of short incubations with anti-cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bionanocapsule-based enzyme-antibody conjugates for enzyme-linked immunosorbent assay

Macromolecules that can assemble a large number of enzyme and antibody molecules have been used frequently for improvement of sensitivities in enzyme-linked immunosorbent assays (ELISAs). We generated bionanocapsules (BNCs) of approximately 30 nm displaying immunoglobulin G (IgG) Fc-binding ZZ domains derived from Staphylococcus aureus protein A (designated as ZZ-BNC). In the conventional ELISA using primary antibody and horseradish peroxidase-labeled secondary antibody for detecting antigen on the solid phase, ZZ-BNCs in the aqueous phase gave an approximately 10-fold higher signal. In Western blot analysis, the mixture of ZZ-BNCs with secondary antibody gave an approximately 50-fold higher signal than that without ZZ-BNCs. These results suggest that a large number of secondary antibody molecules are immobilized on the surface of ZZ-BNCs and attached to antigen, leading to the significant enhancement of sensitivity. In combination with the avidin-biotin complex system, biotinylated ZZ-BNCs showed more significant signal enhancement in ELISA and Western blot analysis. Thus, ZZ-BNC is expected to increase the performance of various conventional immunoassays.     

Tandem conjugation of enzyme and antibody on silica nanoparticle for enzyme immunoassay

We present a new type of enzyme-antibody conjugate that simplifies the labeling procedure and increases the sensitivity of enzyme-linked immunosorbent assay (ELISA). The conjugates were prepared through layer-by-layer immobilization of enzyme and antibody on a silica nanoparticle scaffold. A maximal amount of enzyme was immobilized on the nanoparticle, followed by antibody linkage through Dextran 500. The conjugate could be easily purified from unreacted reagents by simple centrifugations. In comparison with the conventional antibody-enzyme conjugate used in ELISA, which often has one or two enzyme molecules per antibody, the new type of conjugate contained more enzyme molecules per antibody and provided a much higher signal and increased sensitivity. When used in an ELISA detection of the hepatitis B surface antigen (HBsAg), the detection limit was three times lower than that of the commercially available ELISA kit.     

Comparison between indirect enzyme-linked immunosorbent assays for Anaplasma marginale antibodies with recombinant major surface protein 5 and initial body antigens

Indirect enzyme-linked immunosorbent assays (ELISAs) based on recombinant major surface protein 5 (rMSP5) and initial body (IB) antigens from a Brazilian isolate of Anaplasma marginale were developed to detect antibodies against this rickettsia in cattle. Both tests showed the same sensitivity (98.2%) and specificities (100% for rMSP5 and 93.8% for IB ELISA) which did not differ statistically. No cross-reactions were detected with Babesia bigemina antibodies, but 5 (rMSP5 ELISA) to 15% (IB ELISA) of cross-reactions were detected with B. bovis antibodies. However, such difference was not statistically significant. Prevalences of seropositive crossbred beef cattle raised extensively in Miranda county, state of Mato Grosso do Sul, Brazil, were 78.1% by rMSP5 ELISA and 79.7% by IB ELISA. In the analysis of sera from dairy calves naturally-infected with A. marginale, the dynamics of antibody production was very similar between both tests, with maternal antibodies reaching the lowest levels at 15-30 days, followed by an increase in the mean optical densities in both ELISAs, suggesting the development of active immunity against A. marginale. Results showed that all calves were seropositive by one-year old, characterizing a situation of enzootic stability. The similar performances of the ELISAs suggest that both tests can be used in epidemiological surveys for detection of antibodies to A. marginale in cattle.     

Antibody profile in symptomatic/asymptomatic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infected Saudi persons

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected persons could be symptomatic or asymptomatic. Asymptomatic and symptomatic patients can transmit SARS-CoV-2. This study aimed to study the humoral immune response in Saudis who are Covid-19 symptomatic and asymptomatic patients. We created three types of enzyme-linked immunosorbant assays (ELISAs) to reveal IgG and IgM antibodies (Abs) against SARS-CoV-2. The developed ELISAs were designed to detect Abs against SARS-CoV-2 N, S and N + S proteins. A number of Covid-19 symptomatic (1 5 3) and asymptomatic (84) RT–PCR-confirmed patient sera were used to evaluate the ELISAs and to determine the IgG and IgM antibody profile in those patients. The sensitivity and specificity of these ELISAs were evaluated using pre-Covid-19 pandemic serum samples. The results revealed the existence of anti-SARS-CoV-2 IgG and IgM Abs in Covid-19 symptomatic and asymptomatic Saudi persons. The use of SARS-CoV-2 N and S proteins in the same ELISA greatly increased the detectability of infection. In conclusion, the Covid-19 symptomatic and asymptomatic Saudi persons demonstrated both IgG and IgM antibody profile with higher titer in symptomatic patients. The use of N + S proteins as antibody capture antigens greatly increased the ELISA sensitivity.     

Magnetic acoustic resonance immunoassay (MARIA): a multifrequency acoustic approach for the non-labelled detection of biomolecular interactions

A unique sensing platform, comprising an electromagnetic field detector and an acoustic resonator, has been used as a wireless system for remote sensing of biorecognition events. The MARS (Magnetic Acoustic Resonator Sensor) technique has proven useful for detecting the formation of protein multilayers derived from specific binding phenomena. The technique enables multifrequency analysis, without the need of electrodes attached to the sensing element, and also facilitates the in situ surface modification of the substrate for antibody attachment. The MARS sensor was utilized as the platform on which a standard immunoassay was carried out. Two different conditions for the attachment of the first antibody to the quartz surface were tested: (i) Adsorption of the antibody onto the surface of a bare quartz disc; (ii) covalent immobilization of the antibody to a chemically modified quartz surface. Both methods can be successfully utilized for the 'label-less' detection of the biorecognition event between goat IgG and anti-goat IgG by analysis of the multifrequency spectrum. Covalent attachment of the primary antibody results in a more efficient immobilization, with higher surface density, and a consistently enhanced response for the binding of the secondary antibody. This approach will be of interest to life scientists and biochemists that require high performance assay methodologies that do not use chemical labels.     

Novel potentiometry immunoassay with amplified sensitivity for diphtheria antigen based on Nafion, colloidal Ag and polyvinyl butyral as matrixes

A novel potentiometry immunoassay with amplified sensitivity has been developed for the detection of diphtheria antigen (Diph) via immobilizing diphtheria antibody (anti-Diph) on a platinum electrode based on Nafion, colloidal Ag (Ag), and polyvinyl butyral (PVB) as matrixes in this study. The modified procedure was further characterized by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The influence and factors influencing the performance of resulting immunosensor were studied in detail. The resulting immunosensor exhibited sigmoid curve with log Diph concentrations, high sensitivity (51.4 mV/decade), wide linear range from 8 to 800 ng ml(-1) with a detection limit of 1.5 ng ml(-1), rapid potentiometric response (<3 min) and long-term stability (>6 months). Analytical results of clinical samples show that the developed immunoassay is comparable with the enzyme-linked immunosorbent assays (ELISAs) method, implying a promising alternative approach for detecting diphtheria antigen in the clinical diagnosis.     

A rapid procedure for preparing fluorescein-labeled specific antibodies from whole antiserum: its use in analyzing cytoskeletal architecture

A rapid method for the direct conjugation of affinity-purified antibodies with fluorescein (termed DCAPA) is described. This procedure involves the immobilization of antibodies as antigen-antibody complexes on nitrocellulose blots, and subsequently the bound antibodies are reacted with fluorescein isothiocyanate. An enriched sample of smooth muscle tropomysin transferred to nitrocellulose paper by the Western blotting procedure has been used as the affinity medium for purification of specific tropomyosin antibody from whole rabbit antiserum. Direct conjugation of the antibody with fluorescein was carried out following the binding of antibody to antigen. Direct conjugation and affinity purification of antibodies directed against tropomyosin was accomplished in 2-3 d using an enriched tropomyosin sample and whole antiserum directed against tropomyosin. The immunofluorescence images obtained with this procedure exhibit distinct advantages with regard to background fluorescence and overall specificity of antibody binding. The usefulness of this direct conjugation method in various experimental protocols is discussed.     

An enzyme-linked immunosorbent assay (ELISA) for plasma testosterone

A rapid, single extraction ELISA for testosterone in plasma is described, using a standard 96 well microtitre plate. Testosterone is covalently bonded to bovine thyroglobulin and passively adsorbed in guanidine hydrochloride to the ELISA plate, giving an immobilised antigen approach which simplifies subsequent assay standardisation for steroid hormone assays. The addition of standard, sample and first antibody (rabbit anti-testosterone), which is unique for each different assay, is followed by a general procedure which includes washing, addition of peroxidase labelled goat antirabbit IgG, further washing and finally, addition of o-phenylenediamine substrate with colour development and reading of the plate at 492 nm on an automatic ELISA processor. The ELISA assay is compared to a testosterone RIA with 125I-label and has similar specificity and precision to the latter with a quicker processing time, and is more cost effective. The added advantages that ELISA assays confer over RIAs in terms of isotope purchase and disposal make this an ideal procedure for use in a routine steroid laboratory.     

The immunochemistry of solid-phase sandwich enzyme-linked immunosorbent assays

Multivalent antigens (Ags) such as membrane proteins can be quantitated by using sandwich enzyme-linked immunosorbent assays (ELISAs), which typically show sensitivity from 0.1 to 50 ng/ml. The percentage of antigen that binds in the log-log linear region reflects the affinity of the capture antibody (CAb), and the range of linearity for assays conducted with a particular CAb is proportional to the antibody (Ab) concentration. The sandwich ELISA titration plot reflects the actual amount of Ag bound when asymmetrical configurations are used; steric hindrance that occurs with certain symmetrical configurations, especially when enzyme-Ab conjugates of greater than or equal to 10(6) daltons are used, can alter this relationship. Monoclonal CAbs bind less Ag than polyclonal CAbs. Immobilization of monoclonal CAbs by using a modified avidin-biotin system can result in greater antigen capture capacity (AgCC) than when the Abs are directly adsorbed on plastic. Adsorption of proteins on polystyrene is noncovalent and proportional to the amount added for up to 150 ng/200 microliter in a microtiter well. Adsorption can result in substantial loss of antigenic or antibody activity. Desorption is continuous at a low level and can negatively influence the results of an immunoassay. Data from microtiter sandwich ELISAs can be readily acquired and analyzed by using a computer-based analysis system (ELISANALYSIS) written for the IBM PC. This analytical system considers the immunochemical principles of sandwich ELISAs predicted theoretically and demonstrated empirically.     

Stable enzyme biosensors based on chemically synthesized Au-polypyrrole nanocomposites

This work describes development and optimization of a generic method for the immobilization of enzymes in chemically synthesized gold polypyrrole (Au-PPy) nanocomposite and their application in amperometric biosensors. Three enzyme systems have been used as model examples: cytochrome c, glucose oxidase and polyphenol oxidase. The synthesis and deposition of the nanocomposite was first optimized onto a glassy carbon electrode (GCE) and then, the optimum procedure was used for enzyme immobilization and subsequent fabrication of glucose and phenol biosensors. The resulting nanostructured polymer strongly adheres to the surface of the GCE electrode, has uniform distribution and is very stable. The method has proved to be an effective way for stable enzyme attachment while the presence of gold nanoparticles provides enhanced electrochemical activity; it needs very small amounts of pyrrole and enzyme and the Au-PPy matrix avoids enzyme leaking. The preparation conditions, Michaelis-Menten kinetics and analytical performance characteristics of the two biosensors are discussed. Optimization of the experimental parameters was performed with regard to pyrrole concentration, enzyme amount, pH and operating potential. These biosensors resulted in rapid, simple, and accurate measurement of glucose and phenol with high sensitivities (1.089 mA/M glucose and 497.1 mA/M phenol), low detection limits (2 x 10(-6)M glucose and 3 x 10(-8)M phenol) and fast response times (less than 10s). The biosensors showed an excellent operational stability (at least 100 assays) and reproducibility (R.S.D. of 1.36%).